ABSTRACT Syphilis is a devastating human infection caused by Treponoma pallidum (Tp). Within this overall STI CRC application entitled ?Syphilis vaccine to protect against local and disseminated T. pallidum infection?, this Project concerns the CD4 T cell and antibody response to Tp Vaccine Candidate Antigens (TpVCA) identified by Project 1 and Project 2 Leaders. These include Tp0751, an outer membrane protein involved in endothelial cell adhesion, and members of the Tpr family. Collectively, our research team has shown using the rabbit model of Tp vaccination and infection that vaccination with these TpVCA leads to significant, partial protection against challenge. Protection is thought to be antibody mediated. In turn, antibody responses are dependent on CD4 T cell help from T follicular helper (TFH) cells. The TH1 CD4 T cell response also assists Tp-specific antibody by secreting interferon-gamma to activate macrophages to phagocytose Tp organisms coated by specific antibody. The premise of Project 3 is that a detailed understanding of the acquired immune response to TpVCA will assist a reiterative process by which Projects 1 and 2 will be able to improve TpVCA design, Project 3 contains three Aims. In Aim 1, state-of-the-art single B cell technology will be used to isolate nave and memory rabbit and human B cells that produce TpVCA-specific monoclonal antibodies (mAb). These mAbs will be studied in functional assays by Projects 1 and 2 for anti-Tp functional activities such as opsonophagocytosis and Tp neutralization in infection challenge models. The epitopes recognized by the most potent mAbs will be determined, and the crystal structures of mAb-TpVCA complexes determined if possible, to uncover the TpVCA domains that bear functional anti-bacterial epitopes. Aim 2 will rank the TpVCA for prevalence and levels of CD4 TFH and TH1 cells in the blood of persons with symptomatic and asymptomatic Tp infection, and explore whether Tp-specific CD4 T-cells migrate to sites of infection or are retained as tissue resident memory T cells. Aim 3 will test the best candidate TpVCA from Projects 1 and 2, with a series of proprietary adjuvants that are suitable for use in humans, in mouse humoral and CD4 immunogenicity assays. During the STI CRC period, Project 3 will critically synergize with the other Projects and Cores to downselect and optimize antigen and adjuvants with the goal of delivering a lead Tp vaccine candidate suitable for advancement to first in human clinical trials.